255Journal oflmmun" "I{I,'J/,\f",h",¡.. ~3 (1982) 255-260Elsevier Biomedi.:al Pre,.,.

A Novel Method for the Rapid Bleeding of Ratsfrom the Tail Vejn 1

2P.N. Shek and S.A. Howe¡mmun%&)' Group, Biosciences Division, Delence and Civil ¡nstitute 01 Environmental Medicine.

Department 01 Nationa/ Delence. Downsview, Ontario. Canada

(Received 14 January 1982, accepted 17 February 1982)

A method for bleeding rats from the tail vein was developed. Sufficient vasodilation of the tail wasaccomplished by the use of a specially designed warming device. With the help of a physical restrainer, upto 5 mI of blood can be collected from the tail vein and no anaesthesia is r~uired. This bleedingprocedure is CasI, simple to perform, and imposes practically no risk to the animal.

Key words: b!eeding -b/ood -vasodilation -rat -resrrainer -foil vei,r

son of12-25

lntroduction~ '

Blood can be obtained fTom rats by at least 2 techniques: bleeding from theretro-orbitalplexus (Pettit, 1913; Halpern and Pacaud, 1951) and cardiac puncture(Chase, 1967; Campbell et al., 1970; Herbert, 1978). Both methods require anaesthe-tization of the animal before the operation can be performed. Retro-orbital bleedingis impractical on occasions when a relatively large volume of blood is required andlhis approach often renders the animal prone to infection. Cardiac puncture carriesthe risk of a high mortality TaLe, especially when performed by inexperiencedindividuals. In view of these difficulties, we have developed a simple technique inbleeding rats from the tail vein using a specially designed, but simple, tail-warmingdevice. This technique proves to be fast, easy to perform, and capable of providing

up to 5 mI oí blood in a regular bleeding.

1 This is Publication No. 82-P-27 from the Defence and Civillnstitute of Environmental Medicine.2 Correspondence: Dr. PoN. Shek, Immunology Group, Biosciences Division, Defence and Civillnstituteof Environmental Medicine, 1133 Sheppard Ave. W., Downsview, Ontario, M3M 3B9 Canada.

0022-1759/0000-0000/$02.75 @ 1982 Elsevier Biomedical Press

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Matpriais anú Methods

Am~ lalS ale hooded rats weighing about 300 g were purchased from the Charles River

Bre ding Laboratories, Montreal, Quebec. Animals were kept individually in.sta.in-less teel cages and were allowed free access to laboratory rat chow and dnnkmgwat r.

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"""Rest ainerT e rat restrainer was constructed of clear plexiglass (6 mm thick) and its design

is s own in Fig.l. It is basically a cylindrical chamber fitted with 2 perforatedcirc lar end plates. The circular plate at the tail end of the chamber is removable.Eac of the 2 end plates can be adjusted lengthwise to keep the animal properlyrestr ined. A restraining chamber ,,::ith an internal diameter of 5.5 cm is found 1.0 beappr priate for animals weighing in the range of 300-375 g.

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Tailrarming deviceT e tail warmer was made of thermal wires obtained from an electric thermal

blan et (Sunbeam, Brantford, Ont.) coiled closely together to forro an open cylinder8

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Bleeding procedureThe rat to be bled \Vas al!,

the animal exposed through tIthen locked into place to keepcleaned with a cotton swab mset at 46°C, was usedro encltail for a few mmutes was usAfter the warrner was rernov

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Fig. IfDesign of the rat restrainer. Plexiglass cylindrical chamber: thickness. 6 mm; length, 23 cm(~axi um), 13 cm (rninimum); inte~al diamet.er. ~.5 cm; sirle s!ot~ for v~ntilation,.5 crnX6 rnm. Eachclrcul r end plate has a central opemng (6 mm In dlameter) and IS fltted wlth 2 lockmg-screws.

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é~_é "'"~ ~- ---1-- Fig.2. Construction of the tail-war

adhesive tape, the adhesive side facfree ends of the completed theffilal (

as shown in Fig. 2. The therwire around a glass Tod (dian

tapes (Dorninion Tape. Corn'the 2 free ends of the therm,

Willowdale. Ont../ fitted witrI thermal wire w.:re required: length and 1 cm in internal di

DC power suppl.y (Model 6electrical potential in the r

i 41°-48°C wíthin the core of

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Fig.2. Construction of the tail-warming device. (A) A glass rod (1 cm in diameter) was wrapped withadhe~ive tape, the adhesive si de facing out, and the thermal wire was coiled tightly around it. (B) The 2free ends of the completed thermal coil were connected to a second pair of wires fitted with banana plugs.

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as. s~own in Fig. 2.T~e t~~!~~~ ~?il- was assem?!ed simpl~ by looping .the ther~alwlr1 arour:rt~ glass rod (dlameter~icm) and holdmg:thecolls together wlth adheslvetap s (Dominion Tape, Cornwal1, Ont.). The glass rod was then removed and each ofthe 2 free ends of the thermal wire was connected to a hook-up wire (Electrosonic,Wil1owdale, Ont.) fitted with a banana plug at one end. It was found th;:it 3 m oftheJlimal wire were required for making a tail warmer of approximately 16 cm inlen th and 1 cm ininternal diameter. The tail warmer can be connected to a portableDC power supply (Model 6204A, Harrison Lab., N.J.) adjustable to provide ancl~trical po.te~tial in the range ~f 6.5-8.5 V, generating temperatures between41o,-48°C wlthin the core of the tall warmer.

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8Blefding procedure

Ihe rat to be bled was allowed to enter the plexiglass restrainer with the tail oftheanimal exposed through the opening (12 mm) of the circular end plate which wastheh locked into place to keep the animal properly restrained. The tail of the rat wasclefned with a coUon swab moistened with 70% ethanol. The tail warmer, previouslysetat 46°C, was used to enclose the whole length of the tail (Fig. 3). Warming thetai~ for a few minutes was usually found to be adequate for vasodilation to occur.Afier the warmer was removed, the dilated tÍiil veins became visible and a sterile

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22-gauge hypodermic needl-into thc: lu¡n{.;;~ vf onerof 1.(Fig.4). Best insertion was (the needle inserted at an anThe poin1. of insertion was (as the 2 lateral tail veins Wé

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Results and Discussion

Fig. 3~ Application of the raíl warmer for dilation of the tail veins. By appropriate adjustment of the DCp"'\,et supply, the tail-warmcr was set to a predetermined temperature, for example, 46°C. The warmerwas then inserted to enclose the entire length of the anima\'s raíl. After a few minutes, the warmer wasremoted and the tail inspected for visible vasodilation.

Rats may be bled up to athe animal (Campbell el al.,to obtain this quanti.ty of blcadvantage in experimentoS vmethod, unlike that. of carctraumatizing the animars himents where serial blood sa:was desig!1ed to have 2 00]greater restraining efficiencyemphasized that it is very iproper bleedings.

The route via the tail veiinjections (Herbert, 1978). Tby the lack of an efficientsufficient venous retum to 1adequately dilated. The thi(ineffective to apply a chernicfor example, in dilating theartery (Gordon, 1981) of thiing device, sufficient dilatioiuse of a 100 W desk lamp ist.he insertion of the needle.proper entry into the lumenthe vein. the plunger of thethe pulling action should bemately every 0.1 ml of bloopartially collapsedvein befolt.hat following this guideline

Fig.4. Withdrawa] of blood froro lbthe withdrawal of bJ,)(Jd. Aft.er t.hehub of t.he syringe was allowed to r~

pullmg the plunger of lhe syringe jvein. For trouble-fre~ bleeding. it isof fue 2 circular end plates of t.he r

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259

~l.';;gaUgehYPOdermiC needle, connectedto a 5 rnlc'di'sposable'syriiige,waslnséitedinto the urnen oí one oí the distended veins for the steady withdrawal of blood(Fig.4). est insertion was obtained with the bevel of the needle facing upward, andthe need e inserted at an angle of approxirnately 30 along the direction of thevein.The poin of insertion was chosen to be in between 2 ski n scales. The dorsal as wellas the 2 ateral tail veins were found to be appropriate for this bleeding procedure.

Results and Discussion

Rats ~aY be bled up to a volume of 5 mI at a time with no apparent ill effect onthe anim 1 (Campbell et al., 1970). The method described in this report can be usedto obtain t~is quant~ty of blood without hav~ng to anaestheti~e ~he animal?clearly.anadvantag m expenments where anaesthesla would be undeslrable. ThlS bleedingmethod, unlike that of cardiac puncture (Herbert, 1978),--also avoids the risk oftraumatizing the animal's heart and hence is particularly suitable for use in experi-ments where serial blood safi!pling is required. The rat restrainer used in this studywas designed to have 2 continuously adjustable end plates. This design provides

C[:eater restraining efficiency than other devices with lock-step end plates. It must beemphasiz d that it is very important to keep the animal maximally restrained forproper b1 edings.

The r ute via the tail vein of a rat has been used primarily for the purpose ofinjections (Herbert, 1978). The use of the intravenous route for bleeding is hamperedby the 1 k of an efficient vasodilating device for the tail. In order to promotesufficient venous return to the tail for bleeding purposes, the tail veins need to beadequatel dilated. The thick scaly skin of the rat tail renders it impractical andineffectiv to apply a chemical vasodilating agent,like xylene which is very efficient,for exam le, in dilating the marginal veins (Campbell et al., 1970) and the centralartery (G rdon, 1981) of the rabbit ear for bleeding. With the use of the tail-warm-ing devic , st'Jf- ~ent dilation of the tail veins can be achievedwithinminutes. Theuse of a 00 W desk lamp is helpful for the visualization of the veÍns as-weli as fOíthe inserl on of the needle. It was found appropriate to use a 22-gauge needle forproper e try into the lumen of the tail vein. After the needle has been inserted intothe vein, he plunger of the syringe should be pulled back slowly and steadily, andthe pulli action should be stopped momentarily after the withdrawal of approxi~mately e ery 0.1 ml of blood. This approach allows the venous return to refill the

Aaartially ollapsed vein before additional blood is withdrawn. It must be emphasizedWtat folIo ing this guideline is essential for successful and trouble-free bleedings.

Fig. 4. With rawal of blood from the raíl vein. The dorsal as well as the 2 lateral tail veins can be used forthe withdra al of blood. After the needle was inserted into the lumen of the distended vein, the luer-lokhub of the s finge was allowed to test on the thumb of the hand holding the tail.Blood was withdrawn bypulling the lunger of the syringe intermittently, thus allowing the venous return to refill the collapsedvein. For tr uble-fre.e bleeding, it is imperative that the animal be properly restrained by prior adjustmentof the 2 cir lar end plates of the restrainer.

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260I.." \ .

..The kind 01' papers whtCh wi,i'

communicationSc and review-ty¡:present slatus.Submission 01 a paper lo the Jo

previousty been published and 1I

.T~is bleeding method has been used repeatedly (over 100 bleedings) to obtain

,r;~8;;c1?J09~'cf~Qm!ats. Dependingo_~.;t.h~~eed oí. t~_eexp~~~~~~,~"..I":-~"~I,, ~f~lo~d c.1nbecollefted using syringes with or without heparin. Heparinized blood was used for the.f",~""""; prePfration of peripheral blood lymphocytes and non-heparinized blood, for tllecolleftion of serum. To prevent unnecessary haemolysis, the needle of the syringemust: be removed before transferring the collected blood to an appropriate container.The ~se of sterile, disposable plastic syringes (5 mI) was found to be lar moresupe~ior than glass syringeso The tight-fitting design of the plastic syringe cir-cum~ents the problem of the back-flow of blood over the plunger of most glass

syrin~esoT~s bleeding technique should be useful to investigators w~ose experimental

deSiot ay require a relati~ely large volume.of rat blo~d for the ~ollectio~ of nor~alor 1 une sera. By takmg the appropnate aseptIc precautIons, thlS bleedmgproc dure is also useful in obtaining heparinized blood for cell culture studies.

Preparalion o, Ihe lexlThe lenglh of articJes should be I

The manuscript shouJd be accOlTthe original and two copies are r.

(a) Manuscripts. addressed lo orand wide margins. Words to be pi

throughout(b) The title page is supplied astheir affiliations

(c) An abstracto 01 not more than

appear at the beginning 01 the pa

AcknowledgementsKey words

Key words should be provided,

TI1e authors wish to thank Dr. B.H. Sabiston for bis interest and encouragement,Mr. <j;.Jardine and Mr. J. Ulrichsen for their technical assistance in constructing theanimr1 r~strainer .and the t~ warmer, Mrs. l. Boutilier and Ms. D. Gardham for theprep~ratIon of this manuscnpt.

References

Camp~ell, O.H., J.S. Garvey, ~.E. Crerner and 0.1:1. ~ussdorf, 1970, Methods in Imrnunology, ALa~oratoryText for Instructlon and Research (Benjamm, New York) p. 7.

Chasej M.W., 1967, in: Methods in Irnrnunology and Irnrnunochernistry, Vol. 1. eds. C.A. Williarns andH.}v. Chase (Academic Press, New York) p. 254.

Gordqn, L.K., 1981, J. Irnrnunol. Methods 44,241.Halpetn, B.N. and A. Pacaud, 1951, C.R. Seances Soco Biol. Ses Fil. 145, 1465.Herb~.rt, W-I. '1978, in: Handbook of Experimental Irnmunology, Vol. 3, ed. O.W. Weir (Blackwell

Scjentific Publication, Oxford) p. A4.14.Pettit"A., 1913, C.R. Seances Soco Biol. Ses Fil. 74,11.

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